Identification of the compound(s) responsible for the broad-spectrum antimicrobial activity of a bacterium isolated from the indigenous Mexican fermented corn product pozol

Pozol, an indigenous Mexican corn fermentation has a history dating back to the early Mayans. It was used as both a food and medicinally as either a poultice or as treatment for gastrointestinal infections. Bacillus sp. CS93 was previously isolated from pozol and inhibited Gram-positive bacteria, Gram-negative bacteria, yeasts, and molds. This organism could account for pozol's mythical antimicrobial properties. Due to CS93's long history of consumption, the compounds responsible for antimicrobial activity may be useful as natural food preservatives. Therefore, the objective of this study was to identify the compounds responsible for the antimicrobial activity of CS93. Using an ethyl acetate solvent extraction and C-18 reverse phase HPLC, it was discovered that at least three compounds were responsible for the broad inhibitory spectrum of CS93. One of the compounds present in the HPLC fractionation exhibited activity against yeasts and molds. This compound was identified using both analytical reverse phase HPLC and matrix-assisted laser desorption ionization mass spectrometry as iturin A, an antifungal compound produced by a number of Bacillus sp. This HPLC fraction also contained activity against Microccocus luteus. Another HPLC fraction contained activity against Gram negative bacteria. It was inactivated by pronase E suggesting that it was a peptide. The compound was purified using preparative reverse-phase HPLC and size exclusion chromatography. The active fractions from the preparative column were submitted for LC/MS and the data suggested that the compound was bacilysin. This conclusion was supported by two observations. First, pronase E was the only protease to inactivate the compound and this is the only protease to digest bacilysin. Secondly, activity was antagonized by N-acetylglucosamine also known to interfere with the antimicrobial activity of bacilysin. Another compound identified in the same fraction by LC/MS was chlorotetain, which is structurally similar to bacilysin. Bacilysin could have potential for the food industry as a biopreservative.